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Molecular Cancer Research 1:176-185 (2003)
© 2003 American Association for Cancer Research

Cancer Genes and Genomics

Direct Evidence That Bcr-Abl Tyrosine Kinase Activity Disrupts Normal Synergistic Interactions Between Kit Ligand and Cytokines in Primary Primitive Progenitor Cells1

Annabel Strife1, David Wisniewski1, Chongyuan Liu1, Caryl L. Lambek1, Zbigniew Darzynkiewicz4, Richard T. Silver3,5 and Bayard Clarkson1,2,3

1 Molecular Pharmacology and Chemistry Program, Sloan-Kettering Institute and 2 Department of Medicine, Memorial Hospital, Memorial-Sloan-Kettering Cancer Center, New York, NY;
3 Weill Medical College, Cornell University, New York, NY;
4 Cancer Research Institute, New York Medical College, Hawthorne, NY; and
5 New York-Presbyterian Cornell Medical Center, New York, NY

Requests for reprints: Bayard Clarkson, MSKCC Box 96—Room 401C-RRL, 430 East 67th Street, New York, NY 10021. Phones: (212) 639-7490 or 7491; Fax: (212) 717-3053. E-mail: b-clarkson{at}ski.mskcc.org; demerits{at}mskcc.org


   Abstract
Top
 Notes
 Abstract
Introduction
 Results and Discussion
 Materials and Methods
 Acknowledgements
 References
 
We previously reported that chronic myelogenous leukemia (CML) primitive granulocyte-monocyte (GM) progenitors have a greatly reduced requirement for kit ligand (KL) to achieve optimal growth with granulocyte colony-stimulating factor (G-CSF) + granulocyte-monocyte colony-stimulating factor (GM-CSF). Conversely, others have demonstrated that unlike normal, CML CD34+ progenitors can proliferate in response to KL as a sole stimulus. To address these seemingly paradoxical findings, we examined the growth responses of CML CD34+ GM progenitors to various cytokines with and without a potent inhibitor of Bcr-Abl tyrosine kinase activity, PD173955. The heightened growth responses of CML GM progenitors to KL alone and to G-CSF + GM-CSF were abrogated by 10 nM PD173955 while having no effect on normal GM progenitors. While normal GM progenitors exhibited the expected synergistic response when KL was added to G-CSF + GM-CSF, CML GM progenitors had a minimal response; however, some synergism was restored by 10 nM PD173955. Normal erythroid progenitors require the synergistic interaction between KL and a saturating amount of erythropoietin (EPO, 1 unit) for optimal growth. In contrast, CML erythroid progenitors had up to 50% of optimal growth in KL alone, and, only a subthreshold amount of EPO (0.1 unit) was needed with KL to achieve 85% of the optimal response; these heightened growth responses were largely abrogated by 10 nM PD173955. Thus, direct evidence is provided that constitutively activated Bcr-Abl kinase pathways in primitive CML progenitors cooperate with single growth factors producing a heightened growth response, and, in so doing, disrupt the normally required synergistic interactions between KL and other cytokines to achieve activation and optimal growth of primitive progenitors. Coupled with our previous findings that a larger than normal proportion of CML primitive progenitors are at a later stage of maturation, we propose that this disruption of normal synergistic responses leads to increased progenitor recruitment into a committed pool by a process of accelerated maturation.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results and Discussion
 Materials and Methods
 Acknowledgements
 References
 
A reciprocal translocation between chromosomes 9 and 22, resulting in the generation of the chimeric p210bcr-abl activated tyrosine kinase protein, is the hallmark of the chronic phase Ph positive (Ph+) chronic myelogenous leukemia (CML). This specific cytogenetic abnormality [the Philadelphia (Ph) chromosome] is highly consistent in CML and occurs in the pluripotent stem cell compartment (1–9).

During the early chronic phase of CML, the Ph+ cells retain their capacity for relatively normal maturation; nevertheless, there is a greatly increased mass of Ph+ myeloid cells (10). Like their normal counterparts, the Ph+ progenitor cells in the chronic phase of CML are absolutely dependent on growth factors and cytokines for survival, growth, differentiation, and maturation.

Over the past several years, studies in our laboratory which focused on primary CML progenitor cell populations have shown that whereas normal and CML mature granulocyte-monocyte (GM) progenitors have similar growth patterns and similar responses to cytokines, subtle dissimilarities in the phenotypic and growth characteristics of primitive CML progenitors, relative to their normal counterparts, are clearly manifested (11–15). Relative to normal, a greater proportion of enriched CML primitive blasts:

(a) exhibit earlier cytoplasmic maturation;
(b) express the CD33 antigen;
(c) more rapidly lose expression of the CD34 antigen;
(d) have a heightened response to single cytokines (i.e., lower requirement for multiple growth factors);
(e) have a reduced requirement for the synergistic activity of ligand [kit ligand (KL)] with granulocyte colony-stimulating factor (G-CSF) + granulocyte-monocyte colony-stimulating factor (GM-CSF) even though they have similar cell surface expression of the c-kit receptor; and
(f) have less proliferative potential.
Therefore, a greater proportion of CML primitive blasts exhibit characteristics associated with a more advanced stage of maturation than comparable normal blast populations (i.e., the CML primitive progenitor population more closely resembles the normal mature progenitor population). Although there are increased numbers of CML GM and erythroid progenitors, they have less proliferative potential than normal progenitors (11–23). Electron microscopic (EM) studies by Asano and Kawahara (24) and Vainchenker et al. (25) indicated that maturation of earlier CML cells may be asynchronous with cytoplasmic maturation initially more rapid than nuclear maturation. The summation of the evidence from our studies as well as abnormalities reported by others led us to hypothesize that the CML progenitors undergo accelerated maturation at the time of commitment (11–15). Recently, Buckle et al. (26) reported that they have come to a similar conclusion that CML is not a disease of "unbridled stem cell proliferation," but rather the consequence of bcr-abl tyrosine kinase activity in vivo, altering the self-renewal/maturation balance in CML primitive stem cells in favor of maturation.

It is well known that normal primitive GM progenitors require multiple growth factors including KL to achieve optimal cell growth. However, as these normal GM progenitors mature, they lose expression of the kit receptor (27) and become dependent on lineage specific cytokines such as G-CSF for continued growth, survival, and maturation. In light of this, our previous finding that CML primitive GM progenitors have a dramatically reduced requirement for KL suggested that a significantly larger proportion of CML primitive progenitors were at a more advanced stage of maturation.

Moore et al. (28) recently demonstrated that CML CD34+ progenitor cells, unlike their normal counterparts, can proliferate in response to KL as the sole stimulus, thus confirming that kit receptors expressed on CML progenitor cells can be activated by KL. To address this seeming paradox, in the present studies, we examine the growth response of highly enriched CML CD34+ GM (CD13+/CD15+) and erythroid (CD36+) progenitor cells to recombinant hematopoietic growth factors in short-term suspension culture in the absence and presence of a novel, potent pharmacological inhibitor of bcr-abl tyrosine kinase activity, i.e., PD173955. Our results provide direct evidence that the constitutive tyrosine kinase activity of bcr-abl at the time of commitment:

(a) reduces the requirement for multiple growth factors to recruit primitive GM progenitors into a committed pool, and, in so doing, disrupts the normal synergistic activity of KL with G-CSF + GM-CSF;
(b) renders a significant proportion of these primitive progenitor cells responsive to subthreshold concentrations of G-CSF + GM-CSF; and
(c) dramatically heightens the growth response of erythroid progenitor cells to KL as a sole stimulus and cooperates with erythropoietin (EPO) growth factor activated pathways in such a manner as to render erythroid progenitor cells sensitive to subthreshold concentrations of EPO in the presence of KL.
In addition, we provide direct evidence that bcr-abl kinase activity no longer plays a significant role in the growth response of maturing CML GM progenitors to cytokines.


   Results and Discussion
Top
 Notes
 Abstract
 Introduction
 Results and Discussion
Materials and Methods
 Acknowledgements
 References
 
Effects of Tyrosine Kinase Inhibitors (PD173955 and STI-571) on Proliferation of Normal and CML CD34+ GM Progenitors
To directly assess the contribution of the constitutively activated bcr-abl tyrosine kinase activity in the growth response of highly enriched primary CD34+ CML GM progenitor cells to KL, G-CSF, and GM-CSF, ideally, one would have to selectively inactivate the p210 kinase activity within these cells without affecting normal growth factor pathways activated by these growth factors. Although STI-571, a highly publicized tyrosine kinase inhibitor, has been shown to be an effective inhibitor of bcr-abl kinase (29), it has also been shown to be almost equally potent in inhibiting the c-kit receptor tyrosine kinase (30). We have recently reported studies on a more potent inhibitor of bcr-abl tyrosine kinase activity, PD173955 (31, 32). This compound, which is a member of a new class of highly potent tyrosine kinase inhibitors based on the pyrido[2,3-d]pyrimidine core template (33), inhibits p210 tyrosine kinase activity at low nanomolar concentrations (IC50 ~2 nM) both in in vitro kinase and cellular assays. Furthermore, in analyses of cell lines that are dependent on cytokines for growth, we demonstrated that PD173955 inhibited KL-dependent proliferation at an IC50 of ~50 nM and GM-CSF-dependent cell growth at an IC50 ~1 µM. Thus, in cellular assays, PD173955 is approximately 25- and 500-fold more inhibitory to Bcr-Abl kinase versus c-kit receptor activated tyrosine kinase and kinases activated by GM-CSF stimulated growth factor pathways, respectively. Therefore, PD173955 was an attractive candidate for the present studies.

We initially examined the effects of various concentrations of PD173955 on the short-term growth of primary normal CD34+ GM progenitors in response to G-CSF + GM-CSF and G-CSF + GM-CSF + KL and compared its effects to that of STI-571. The first objective was to ascertain the highest (threshold) concentration of drug which would leave normal progenitors unaffected; determining this concentration was essential so that any observed inhibitory effects of PD173955 on the growth of comparable primary CML CD34+ GM progenitors at concentrations at or below this threshold would be attributed to the direct inhibition of bcr-abl kinase activity. It can be seen in Fig. 1A that normal CD34+ GM progenitors can be grown in up to 25 nM of either STI-571 or PD173955 with no inhibition. In contrast, 25 nM PD173955 caused pronounced inhibition ({cong}70%) of [3H]thymidine uptake in CML CD34+ GM progenitor cells grown in GM-CSF + G-CSF (Fig. 1B). Because 25 nM PD173955 had no effect on the growth of comparable normal GM progenitors, these results provide direct evidence that the inhibition by 25 nM PD173955 is due to the selective inhibition of the bcr-abl tyrosine kinase activity. In fact, Fig. 1B shows that as little as 10 nM of PD173955 results in the near maximal inhibition (i.e., 25 nM) of CML GM progenitor cell growth. Therefore, a concentration of 10 nM PD173955 was used for all subsequent studies. Cell cycle analysis (Fig. 1C) of CML GM progenitors grown in G-CSF + GM-CSF showed that 10 nM of PD173955 reduced the percentage of cells in S-G2-M (control 26% versus PD173955 15%) while increasing the percentage of cells in G1 (control 74% versus PD173955 85%). It can also be seen in Fig. 1 that a concentration of 250 nM for STI-571 is needed to get about the same degree of inhibition as that obtained with 10 nM PD173955. Although this concentration had minimal inhibitory effects on comparable normal GM progenitors grown in G-CSF + GM-CSF, 250 nM STI-571 significantly inhibited (~40%) [3H]thymidine uptake by normal progenitors grown in KL + G-CSF + GM-CSF and therefore, precluded its use in the present studies.



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  		FIGURE 1. Effect of tyrosine kinase inhibitors PD173955 and STI-571 on DNA synthesis of primary normal and CML CD34+ GM progenitor cells. A. Normal GM progenitors were cultured for 72 h in G-CSF + GM-CSF (10 ng/ml each) with and without 50 ng/ml KL in the presence of various concentrations of PD173955 or STI-571. [3H]thymidine (1 µCi/well) was added for the last 18 h. B. CML GM progenitors were incubated for 72 h in G-CSF + GM-CSF (10 ng/ml each) with various concentrations of PD173955 or STI-571. [3H]thymidine was added for the last 18 h. For both A and B, values obtained in untreated samples are considered as 100%. C. CML GM progenitors that were grown for 72 h in G-CSF + GM-CSF in the absence and presence of 10 nM PD173955 were analyzed for cell-cycle status using propidium iodide staining (see "Materials and Methods"). Results in A, B, and C are representative of two independent experiments.

 
Although not shown in Fig. 1, it is important to emphasize that there is essentially no growth of either normal or CML GM progenitor cells in the complete absence of growth factors, which indicates that the constitutive bcr-abl kinase activity within CML GM progenitor cells cannot replace the absolute requirement for growth factors and cytokines. At first glance, it appears somewhat counterintuitive that when bcr-abl kinase activity is selectively inhibited, a proportion of CML progenitor cells are somehow inhibited in their growth response to saturating concentrations of G-CSF + GM-CSF. We believe that the explanation for this will become apparent in the findings described below.

PD173955 (10 nM) Inhibits Global Bcr-Abl-Dependent Tyrosine Phosphorylation in CD34+ Stem Cells From Primary CML Cells in Vivo
We have previously demonstrated a strikingly consistent pattern of proteins constitutively phosphorylated on tyrosine in primary CML progenitor cells that is not observed in comparable primary normal progenitor cells (34–37). To demonstrate that PD173955 directly inhibits the autophosphorylation of the bcr-abl tyrosine kinase in vivo, primary CML CD34+ progenitor cells were incubated in G-CSF + GM-CSF overnight in the presence and absence of 10 nM of PD173955. It is shown in Fig. 2A that 10 nM PD173955 dramatically inhibited the autophosphorylation of the p210bcr-abl protein as well as its protein substrates that we have previously identified in primary CD34+ CML progenitors as SHIP1, SHIP2, Cbl, and p62dok. That the observed dephosphorylation shown in Fig. 2A was not due to a reduction in the amount of each of these proteins was demonstrated by stripping the blot in Fig. 2A and reprobing with antibodies specific for the proteins shown in Fig. 2B. Thus, 10 nM PD173955 directly inhibits bcr-abl kinase activity in primary CD34+ CML progenitor cells.



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  		FIGURE 2. PD173955 inhibits global Bcr-Abl-dependent tyrosine phosphorylation in primary CML CD34+ progenitor cells in vivo. Freshly isolated CML CD34+ progenitor cells were incubated for 24 h with G-CSF + GM-CSF in the absence and presence of 10 nM PD173955. Cells were harvested, rinsed in PBS and lysed in hot SDS sample buffer. Cell lysates (1.7 x 106 cell equivalents) were separated by 7.5% SDS-PAGE, transferred to an Immobilon membrane and immunoblotted with an anti-ptyr MoAb (A). Anti-ptyr blot was stripped and reprobed sequentially with antibodies specific to Abl, SHIP2, SHIP1, Cbl, and p62dok proteins (B). Results shown are representative of two separate experiments.

 
Comparison of the Growth Response of Normal and CML GM Progenitor Cells to KL, GM-CSF, and G-CSF in the Presence and Absence of PD173955
Fig. 3A shows that CML GM progenitors (compared to normal) display a marked increase in their response to KL as a sole stimulus. While normal enriched GM progenitors in the presence of KL exhibited only ~17% (three normal donors range 16–18%) of the maximal proliferative response (seen in KL + G-CSF + GM-CSF), CML GM progenitors exhibited ~44% (eight CML patients range 32–50%) of the maximal proliferative response. Moreover, 10 nM PD173955 completely abrogated this heightened response to KL and returned the [3H]thymidine uptake values to those seen with normal GM progenitors with KL as the sole stimulus.



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  		FIGURE 3. Comparison of growth response between CML and normal CD34+ GM progenitors to either KL, G-CSF + GM-CSF or KL + G-CSF + GM-CSF in the absence and presence of 10 nM PD173955. A. Normal or CML progenitors were incubated for 72 h in the various growth factors at concentrations indicated without and with 10 nM PD173955. [3H]thymidine (1 µCi/well) was added for the last 18 h. Data (mean value, see text for ranges) are normalized. Each CML patient and each normal donor progenitor population was individually normalized to 100% of the value obtained with their population in the presence of G + GM + KL which was considered the maximum response. {square}, Untreated; {blacksquare}, PD173955. B. Aliquots of CML and normal GM progenitors that were grown for 72 h in saturating amounts of G-CSF + GM-CSF without or with KL (50 ng/ml) in the absence or presence of 10 nM PD173955 (CML progenitors only) were analyzed for cell-cycle status using propidium iodide staining.

 
To determine whether this increased uptake of [3H]thymidine by CML GM progenitors in KL reflects an actual growth response or merely an increased survival of cells initially in cycle, CML and normal GM progenitors were cultured in serum-free medium (SFM) supplemented with KL at very low cell concentrations (1000 cells/well) and the viable cell number was assessed after 6 days (Fig. 4). Under these conditions, the CML cell number increased ~1.6-fold, whereas the normal cell number decreased to 25% of the initial starting cellular concentration. However, in the presence of PD173955, CML cell expansion was completely abrogated. These results provide direct evidence that bcr-abl kinase activity in CML GM progenitor cells cooperates with kit kinase activated pathways when KL is the sole stimulus.



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  		FIGURE 4. Growth response of CML and normal CD34+ GM progenitors to KL at Day 6. Progenitor cells (initially placed at 1000/well) were cultured in serum-free medium supplemented with KL (100 ng/ml) in the absence and presence of 10 nM PD173955. The number of viable (trypan blue excluding) cells in each well was enumerated after 6 days. Results shown are representative of two separate experiments.

 
Second, the results given in Fig. 3A also show that CML GM progenitors exhibit a heightened responsiveness to either saturating amounts of G-CSF + GM-CSF [CML ~77% (nine CML patients range 45–94%) versus normal ~41% (three normal donors range 29–48%) of maximal response] or subthreshold amounts of G-CSF + GM-CSF [CML ~48% (seven CML patients range 34–67%) versus normal ~17% (two normal donors range 16% and 17%) of maximal response]. This increased responsiveness is directly attributable to the cooperation of bcr-abl kinase activity with G-CSF + GM-CSF activated growth factor pathways because 10 nM PD173955 completely ablated the elevated response to these growth factors and returned the [3H]thymidine incorporation values to those seen with normal GM progenitors grown under the same conditions. Of note, our observation that CML primary primitive GM progenitors are more sensitive to subthreshold amounts of G-CSF + GM-CSF is consistent with the previous study of Gishizky and Witte (38) showing that the growth response of murine bone marrow progenitor cells infected with a retrovirus carrying bcr-abl was significantly more sensitive to subthreshold amounts of growth factors than mock-infected cells.

Third, Fig. 3A shows that the addition of KL to saturating amounts of G-CSF + GM-CSF leads to the growth enhancement of normal GM progenitors in a synergistic manner ([3H]thymidine incorporation was increased an average of ~2-fold over the additive effects of KL alone plus [G-CSF + GM-CSF] alone). This synergism can also be seen in terms of cell cycle analysis (Fig. 3B). The addition of KL to normal GM progenitors growing in G-CSF + GM-CSF increased the S-G2-M fraction from 21% to 34%. In contrast, the addition of KL to saturating amounts of G-CSF + GM-CSF did not lead to a synergistic response in CML GM progenitors because their growth response in the presence of all three factors was significantly less than additive, and in fact, CML GM progenitors exhibited near optimal growth (~77% of maximum) in G-CSF + GM-CSF alone. Furthermore, cell cycle analysis (Fig. 3B) revealed no increase in the fraction in S-G2-M when KL was added to G-CSF + GM-CSF. This dramatically reduced requirement for the addition of KL to achieve optimal growth of CML GM progenitors is consistent with our previous findings (14).

The minimal growth enhancement of CML GM progenitors by KL in the presence of the cytokines G-CSF + GM-CSF at first appears paradoxical in light of the fact that these progenitors exhibited a marked response (~44% of maximum growth) when KL was used as the sole stimulus. The most plausible explanation for this lies in our demonstration that activated bcr-abl kinase in GM progenitors cooperates with either KL activated pathways or G-CSF + GM-CSF activated pathways to induce a heightened growth response. Consequently, this ability of activated bcr-abl kinase to cooperate with single growth factors reduces and disrupts the requirement for synergy between these growth factors that is necessary for normal primitive GM progenitors to achieve optimal growth. Evidence that such is the case is the observation (Fig. 3) that the inhibition of growth of CML GM progenitors by 10 nM PD173955 in the presence of all three growth factors [~30% (seven CML patients range 13–56%)] was significantly less than in the presence of KL alone [~77% (seven CML patients range 60–92%)] or G-CSF + GM-CSF alone [~57% (eight CML patients range 40–73%)]. Because we have shown that 10 nM PD173955 potently and efficiently inhibits bcr-abl kinase activity in CML GM progenitor cells, this result therefore indicates that some degree of synergy has been restored. Results of cell cycle analysis (Fig. 3B) also support this conclusion. Treatment of CML GM progenitors with 10 nM PD173955 in the absence of KL reduced the S-G2-M fraction from 33% to 22%, whereas the addition of KL restored the S-G2-M fraction to 29%.

The Effects of Inhibition of Bcr-Abl Kinase Activity Are Diminished in More Mature CML GM Progenitors
Our data thus far indicate that activated bcr-abl kinase within CML GM progenitor cells reduces the requirement for multiple growth factors (Fig. 3) that are normally needed to induce a primitive GM progenitor to enter a commitment pool (G0-G1->S). This would suggest that as CML (as well as normal) GM progenitors mature and subsequently no longer require the presence of multiple growth factors to achieve optimal growth (14, 39, 40), the cooperative effects of bcr-abl kinase activity with growth factor(s) would be diminished in more mature CML GM progenitors.

To provide direct evidence that such is the case, we incubated freshly isolated CML CD34+ GM progenitors for 4 days in the presence of G-CSF + GM-CSF to allow some degree of maturation to occur. These cells were subsequently washed and resuspended in the various growth factors in the presence and absence of PD173955 and examined for [3H]thymidine uptake. The results were compared to those obtained with the respective CML CD34+ GM progenitors initially assayed. In addition, normal CD34+ GM progenitors were grown and assayed under the same conditions. Phenotypic analysis confirmed that both CML and normal GM progenitors were more mature after 4 days in the presence of G-CSF + GM-CSF because the expression of the CD34 antigen was significantly reduced and expression of CD33, CD13, 14, and 15 increased (data not shown).

The results shown in Fig. 5C and D demonstrate that the inhibitory effect of 10 nM PD173955 on CML CD34+ GM progenitors grown in G-CSF + GM-CSF (0–5 days drug added initially) was substantially more pronounced than on CML maturing GM progenitors (4–8 days drug added at 4 days) (inhibition 45% versus 15%, respectively). This result provides evidence that as CML GM progenitors mature, they become much less dependent on the intrinsic bcr-abl kinase activity to optimally respond to G-CSF + GM-CSF. To show that 10 nM PD173955 was effective in inhibiting bcr-abl tyrosine kinase activity in these more mature GM progenitors, protein lysates were obtained after overnight incubation of these cells in G-CSF + GM-CSF in the presence and absence of 10 nM PD173955. As shown in Fig. 6, constitutive bcr-abl tyrosine kinase activity is quite evident in CML cells following 4 days of culture and 10 nM PD173955 was very efficient at inhibiting this activity. We should point out that, although the blot depicted in Fig. 6 does not show the presence of the p210 ptyr bcr-abl protein, longer exposure times detected its presence (data not shown). This is in contrast to initial CML CD34+ progenitor cells from the same patient where the p210bcr-abl protein is readily detectable (see Fig. 2). Although blotting with an ABL antibody was not performed on the blot depicted in Fig. 6, we have observed in other experiments that the expression level of BCR/ABL protein decreases with time in culture (data not shown). This observation is consistent with the previous findings of others (41).



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  		FIGURE 5. Comparative analysis of growth responses of normal and CML CD34+ GM progenitor cells and their more mature progeny in the absence and presence of 10 nM PD173955. (A) Normal and (C) CML CD34+ GM progenitors were incubated for 120 h in the various growth factors indicated without or with 10 nM PD173955. [3H]thymidine was added for the last 18 h. Initial normal and CML CD34+ GM progenitors were grown for 4 days in G-CSF + GM-CSF to allow for some degree of maturation. These cells were then harvested, washed, and incubated for an additional 96 h in the various growth factor conditions indicated (B and D). [3H]thymidine was added for the last 18 h. Data are normalized versus maximal responses (to KL + G-CSF + GM-CSF) (see legend for Fig. 3) and values are representative of three independent experiments. {square}, Untreated; {blacksquare}, PD173955.

 


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  		FIGURE 6. PD173955 inhibits global Bcr-Abl-dependent tyrosine phosphorylation in CML GM progenitor cells that have progressed to a later stage of maturation. Freshly isolated CML CD34+ GM progenitor cells were placed in liquid culture for 96 h with G-CSF + GM-CSF (to induce proliferation and maturation) before adding PD173955 (10 nM final concentration) and cultured for an additional 24 h in cytokines. Cells (0.8 x 106) were harvested, rinsed in PBS, and lysed in hot sample buffer. Cell lysates were resolved by SDS-PAGE, transferred to Immobilon, and immunoblotted with an anti-ptyr MoAb. Results shown are representative of two independent experiments.

 
In looking at the growth response of normal GM progenitors to the various growth factors (Fig. 5A and B), the requirement for the synergistic activities of KL, seen for more primitive normal progenitors (0–5 days) (Fig. 5A), is largely alleviated in normal maturing GM progenitors (4–8 days) (Fig. 5B). Importantly, comparison of Fig. 5B and D reveals that the growth response of maturing CML GM progenitors to either G-CSF + GM-CSF or KL alone is now very similar to what is observed for maturing normal GM progenitors and PD173955 has very little inhibitory effect on the growth of maturing CML GM progenitors, which indicates that the role of bcr-abl kinase activity diminishes in maturing CML GM progenitors.

Bcr-Abl Kinase Activity Alleviates the Normal Requirement for the Synergistic Activities Between KL and EPO in CML Erythroid Progenitor Cells
We also investigated whether intrinsic bcr-abl kinase activity could reduce the well-known normal requirement for the synergistic interaction(s) between KL and EPO for optimal growth of erythroid progenitor cells (15, 42). Thus, enriched CML erythroid progenitors were examined for their growth response in short-term serum-free suspension culture with either KL alone, or varying concentrations of EPO alone, or KL together with varying concentrations of EPO and with and without 10 nM PD173955. Comparable normal erythroid progenitors, used as a positive control, were assayed under the same conditions. The results are shown in Fig. 7.



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  		FIGURE 7. Growth response of normal and CML erythroid progenitors to KL and/or EPO. Normal and CML-enriched CD34+ erythroid progenitors were cultured for 72 h in serum-free medium with either KL alone (100 ng/ml) or various concentrations (indicated) of EPO alone or KL (50 ng/ml) + various concentrations of EPO. [3H]thymidine (1 µCi/well) was added for the last 18 h. Data are normalized versus maximal response (KL + 1 unit EPO). Each CML patient and each normal donor progenitor population was individually normalized to 100% of the value obtained with their population in the presence of KL + 1 unit EPO which was considered the maximum response. Similar results were obtained in two other separate experiments. {square}, Untreated; {blacksquare}, PD173955.

 
The presence of both KL and EPO is essential for any appreciable growth response for normal erythroid progenitors. This growth response is due to a remarkable synergistic interaction between KL and EPO because neither factor elicited any significant response (Fig. 7, top panel). Furthermore, the contribution of EPO in this synergistic response is concentration dependent. In stark contrast, CML erythroid progenitors achieved as much as 50% of the maximal growth response (KL + EPO 1 unit) with KL as the sole factor (Fig. 7, bottom panel). Moreover, a suboptimal concentration of EPO (0.1 unit) in combination with KL achieved 85% of an optimal response.

As is the case with CML CD34+ GM progenitors shown in Fig. 3, the exaggerated response of CML CD34+ erythroid progenitors to KL and EPO as single factors can be directly attributed to the cooperativity of bcr-abl kinase activity with these factors because 10 nM PD173955 completely abrogated the heightened response and returned the [3H]thymidine uptake values to those seen with normal erythroid progenitors. Moreover, 10 nM PD173955 partially restored the normal synergistic response in the presence of EPO 1 unit + KL.

Thus, our findings show that the constitutive activity of bcr-abl present in CML erythroid progenitor cells greatly reduces the normal absolute requirement for the synergistic activities between KL and EPO to achieve optimal growth of erythroid progenitor cells. Thus, CML erythroid progenitor cells (compared to normal) also exhibit a dramatically heightened response to KL as a single agent. This observation is consistent with previous human and murine studies, respectively (43, 44), demonstrating that hemoglobinized colonies could be generated from CML CD34+ cells with KL only. In addition, we demonstrate that bcr-abl kinase activity can cooperate with EPO activated pathways in such a manner as to render CML erythroid progenitor cells more sensitive to subthreshold concentrations of EPO.

Summary
In summary, our data suggest that the substantially heightened growth response of primitive CML progenitors to a single growth factor facilitates an increased recruitment into the committed progenitor pool culminating in the known excessive accumulation of both GM and erythroid CML progenitors. However, as pointed out in the introduction, we (as well as others) (11–15, 45), have previously obtained biological, phenotypic, and biochemical evidence clearly indicating that a substantially larger proportion of CML GM and erythroid progenitors are at a later stage of maturation than their normal counterparts and consequently have, overall, less proliferative capacity. Taken together, therefore, we further propose that the bcr-abl kinase pathways that are constitutively activated in CML primitive progenitors cooperate with growth factors to increase the number of primitive stem cells that are activated (G0-G1->S) and hence recruited to enter the committed progenitor pool. The cooperative interactions between bcr-abl and cytokine-activated pathways disrupt the synergistic interactions between multiple growth factors that are normally required for this process, tipping the balance between proliferative capacity and maturation in favor of maturation. Thus, the CML progenitors that are recruited into the committed pool exhibit accelerated maturation and reduced proliferative capacity in comparison to normal committed progenitors.

Further evidence to support this concept comes from the recent observations of Buckle et al. (26) which demonstrated by multiparameter flow cytometric analysis that the most primitive stem cells were present as a lower proportion of the stem cell compartment of CML patients. Conversely, there was a significantly higher proportion of more mature cells in the stem cell compartment in CML patients than in normal individuals. Of particular relevance to this are the previous studies by other investigators (39, 40, 42) providing biological evidence that synergism between KL and other cytokines results in a delay in differentiation among the progeny of individual progenitor cells while greatly increasing the proliferation of these progeny. Collectively, our studies and those of other investigators suggest that the bcr-abl tyrosine kinase disrupts the normal synergistic response and as a consequence brings about increased progenitor cell recruitment into a committed pool coupled with a process of accelerated maturation.

It will thus be important to begin to search for those downstream signaling pathways affected by the aberrant Bcr-Abl tyrosine kinase activity in primary CML progenitor cells. Although activation of numerous pathways (e.g., Ras, PI-3 kinase, Jak/Stat, and MAPK) is observed in Bcr-Abl transformed myeloid cell lines (49), the relevance of these findings to the abnormal myeloid expansion that is manifested in primary CML progenitor cells has not been established. Future studies from our laboratory will initially focus on the phosphorylation status (which reflects activation) of MAPK, STAT5, JAK2, and AKT proteins in CML progenitor cells constitutively and following culture in the various growth factors in the absence and presence of PD173955. It is anticipated that such studies will help to identify key growth factor signaling pathways that are affected by the presence of Bcr-Abl tyrosine kinase.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
Acknowledgements
 References
 
Hematopoietic Growth Factors
Human recombinant stem cell factor (SCF) or KL, G-CSF, and GM-CSF were gifts from the Kirin Brewery Co., Gunma, Japan. Unless otherwise stated, KL was used at a final concentration of 50–100 ng/ml, whereas G-CSF and GM-CSF were used at subthreshold (0.03 ng/ml each) and optimal (10 ng/ml each) concentrations.

Tyrosine Kinase Inhibitors
PD173955 was initially provided by Parke-Davis Pharmaceutical Research (Division of Warner-Lambert Co., Ann Arbor, MI) and subsequently synthesized as described (33) in the Core Preparative Synthesis Lab at Memorial Sloan-Kettering Cancer Center (MSKCC). Stock solutions at 5 mM were prepared with DMSO and stored at -20°C.

STI-571 or imatinib mesylate (Gleevec, formerly CGP57148B) was initially provided by Dr. Nicholas Lydon of Novartis Inc. (Basel, Switzerland) and also subsequently synthesized as described (29) at MSKCC. Stock solutions at 10 mM were prepared in absolute ethanol and stored at -20°C. For both compounds, fresh working solutions were prepared in Iscove's modified Dulbecco's medium (IMDM) just before use.

Human Peripheral Blood and Bone Marrow Specimens
Fresh normal bone marrow mononuclear cells (MNC) were purchased from Poietic Technologies, Inc. (Bio Whittaker Co., Gaithersburg, MD) and used directly to isolate CD34+ GM or erythroid enriched progenitor cells. Marrow aspirations or peripheral blood specimens were obtained from chronic phase CML patients. Buffy coat cells were depleted of platelets and light-density cells were obtained and cryopreserved as previously described (46). After thawing, cells were suspended in IMDM containing 20% FCS (Hyclone Laboratories, Logan, UT) and DNase 160 units/ml (Worthington Biochemical Corp., Lakewood, NJ). Dead cells were removed by Ficoll-Hypaque centrifugation. Viable MNC were washed in IMDM + 20% FCS and processed for enrichment of CD34+ GM or erythroid progenitor cells.

Enrichment of CD34+ Progenitor Cells with a GM Phenotype (CD13+/CD15+)
Fresh light-density normal bone marrow (BM) MNC or thawed CML BM or peripheral blood MNC were incubated with CD36, CD41, CD61, and antiglycophorin monoclonal antibodies (Mabs) (Beckman Coulter, Brea, CA) for 30 min on ice, washed twice, and incubated with sheep antimouse IgG conjugated magnetic beads (DYNA beads, Dynal, Oslo, Norway) for 30 min. The antibody-positive cells were removed using a magnetic particle concentrator (Dynal). CD34+ cells were then positively selected using the MACS CD34 isolation kit (Miltenyi Biotec, Inc., Auburn, CA) according to the manufacturer's instructions. The final cell population (as assessed by FACScanTM, BD Immunocytometry Systems, San Jose, CA) was typically >95% CD34+, 40–80% CD13+/CD15+, 40–70% c-kit+ and essentially devoid of cells committed to erythroid (CD36+, glycophorin A+) and megakaryocytic (CD41, CD61) lineage.

Enrichment of CD34+ Erythroid Progenitor Cells
For the negative selection of enriched CD34+ erythroid progenitor cells, cells were incubated with CD13, CD14 (monocytic), CD15, CD41, CD61, CD19 (B cell) Mabs (Beckman Coulter) and the antibody-positive cells were removed using a magnetic particle concentrator (Dynal). The procedure was the same as described for enrichment of CD34+ GM progenitor cells.

Measurement of DNA Synthesis
The proliferative response of normal and CML CD34+ GM or erythroid progenitor cells to growth factors was assessed by measuring the rate of [3H]thymidine incorporation into DNA. Cells (1–4 x 104/well) in 96-well plates were incubated at 37°C in a humidified atmosphere of 5% CO2 in 0.2 ml of IMDM + 5% FCS or QB SF-60 serum-free medium (Quality Biological, Inc., Gaithersburg, MD) containing various growth factors in the presence and absence of tyrosine kinase inhibitors for 48–96 h followed by further incubation with 1 µCi of [3H]thymidine 6.7 Ci/mmol (New England Nuclear, Boston, MA) for 18 h. Cells were then harvested using a Packard Filter Mate harvester (Packard BioScience Co., Meriden, CT) and total counts measured using a Packard TopCount NXT microplate detection system. Values are expressed as counts/min and represent the mean of three replicates.

Suspension Cultures
To assess the potential effects that tyrosine kinase inhibitors may have on the growth of CD34+ GM progenitors, cells were suspended at 0.25 x 106/ml in IMDM + 5% FCS containing growth factors with and without tyrosine kinase inhibitors. Cells were maintained in a humidified atmosphere of 5% CO2 at 37°C for 4–6 days and aliquots were removed for viable cell number (as assessed by trypan blue) and cell cycle analysis.

For suspension culture experiments comparing the ability of normal and CML enriched GM progenitors to grow in response to KL as the sole stimulus, cells were suspended at 1 x 103 cells/ml in QB SF-60 for 4–6 days and viable cell number was determined. In some instances, morphological analysis was done by May-Grunwald-Giemsa staining of cytocentrifuge smears.

Cell Cycle Analysis
The cells, initially suspended in 70% ethanol, were centrifuged (200 x g, 4 min) and the cell pellet resuspended in PBS. The cells were then cytocentrifuged onto microscope slides and stained with a solution containing 10 µg/ml of propidium iodide (PI; Molecular Probes, Eugene, OR) and 0.1 mg/ml of RNase A (Sigma Chemical Co., St. Louis, MO) for 30 min at room temperature, as described by Li and Darzynkiewicz (47). These specimens were subjected to DNA content analysis by measurement of cell fluorescence.

Cell fluorescence was then measured by laser scanning cytometer (CompuCyte, Cambridge, MA) using excitation with 488 nm laser line and measuring integral and maximal pixel intensities of green (530±20 nm) and red (>590 nm) fluorescence using the standard settings of filters and dichroic mirrors of LSC. At least 3000 cells were measured per sample (48).

Western Blot Analysis
CML CD34+ GM progenitors were incubated overnight at 37°C (5% CO2) in IMDM (+5% FCS) containing G-CSF + GM-CSF growth factors (as noted in "Results and Discussion") with or without 10 nM PD173955 or 250 nM STI-571. Equivalent numbers of untreated and treated cells (0.25–1 x 106) were then harvested, quickly washed twice with ice-cold IMDM, once with ice-cold PBS, and lysed in hot Laemmli sample buffer [2% SDS, 100 mM DTT, 10% glycerol, 0.01% bromophenol blue, 60 mM Tris-HCl (pH 6.8)] for 5 min at 100°C. Protein lysates were resolved on a 7.5% SDS-polyacrylamide gel, transferred to a polyvinylidene difluoride membrane and immunoblotted with anti-ptyr MoAb 4G10.

Antibodies were stripped from the filters by constant agitation in a solution containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 100 mM ß-mercaptoethanol for 30 min at 55°C. The filters were then washed extensively with Tris-buffered saline containing 0.1% Tween 20 (TBST) and reprobed with different primary antibodies as noted in "Results and Discussion".


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Pfizer Global Research and Development (Ann Arbor Laboratories, MI), formerly Parke-Davis Pharmaceutical Research (Division of the Warner-Lambert Co., Ann Arbor, MI) and Dr. Neal Rosen for initially providing PD173955; and Dr. Nicholas Lydon of Novartis Inc. (Basel, Switzerland) for initially providing STI-571 (formerly known as CGP57148B); and the Kirin Brewery Company, Limited (Tokyo, Japan) for their generosity in supplying rhGM-CSF, rhG-CSF, and rhKL. We also thank Drs. William G. Bornmann, Ephraim Vidal, and Darren Veach at MSKCC's Preparative Synthesis Laboratory for synthesizing compounds and for helpful discussions. We are grateful to Dr. Richard T. Silver's nursing and office staff for their assistance in obtaining samples of CML blood and marrow, especially Eugenie Balam. We thank Su De Meritt for help in preparing the manuscript.


   Notes
Top
 Notes
Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 National Cancer Institute (NCI) Grant CA64593 and NCI Cancer Center Support Grant CA08748, The Albert C. Bostwick Foundation, The Enid A. Haupt Charitable Trust, The Andrew Sage Trust, The Einard and Sue Sundin Fund, The United Leukemia Fund, The Carley H. Wagner Trust, The Westvaco Corporation, and MeadWestvaco. Back

Received July 5, 2002; revised November 8, 2002; accepted November 20, 2002.


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 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 Acknowledgements
 References
 

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